Actinobacillus pleuropneumonia (thereafter “App”) is a Gram-negative bacteria which causes porcine pleuropneumonia, a worldwide distributed infectious disease responsible of important Economic losses in the swine industry.
The App most important virulence factors are extracellular proteins namely: Apx exotoxins. These exotoxins belong to the pore-forming RTX toxins family, widely spread among pathogenic gram-negative bacteria The main exotoxins in App are: ApxI, ApxII, ApxIII and ApxIV.
Exotoxins ApxI and ApxII are haemolytic and cytolytic. ApxI shows a strong haemolytic and cytolytic activity and ApxII shows a weak haemolytic and a moderate cytolytic activity.
Although all, so far screened, serotypes are able to produce ApxIV, there is a characteristic serotype distribution for the expression of the rest of the Apx exotoxins. Serotypes 1, 5, 9 and 11 produce exotoxins ApxI and ApxII; Serotype 10 only produces ApxI; Serotypes 7 and 12 produce only ApxII and serotypes 2, 3, 4, 6, and 8 produce ApxII and ApxIII.
The genes corresponding to exotoxins ApxI and ApxII are organized as operons. The operon of of the ApxI exotoxin contains 4 genes: apxIC, apxIA, apxIB and apxID. The gene of apxIA codes for the ApxI exotoxin itself The gene apxIC codes for an activator protein (acylase) which introduces a post-translational modification (acylation) in the Apx, which allows the ApxI to acquire an active conformation, making it able for the interaction with the host specific cell receptors. The apxIB and apxID genes code two membrane proteins which secrete the mature ApxI exotoxin to the external medium.
The ApxII operon contains only the gene A (apxIIA) and gene C (apxIIC) which code, respectively, for ApxII and for the acylase responsible for the ApxII to acquire an active conformation. There exists also a small fragment which shows a certain similarity with the apxIB gene but it does not originate a functional protein. The export of the mature ApxII to the external medium is due to the action of the proteins encoded by the apxIB and apxID genes.
The present vaccination methods do not provide a complete protection against all App serotypes.
Patent WO97/16532A1 describes the construction of a vaccine strain able to induce an immunological response in an animal. This comprises a modified microorganism which produces a partially or totally inactivated Apx toxin, due to a partial deletion. This deletion, done by induced mutagenesis of the structural gene apxIA and/or to the partial deletion of an apxIIC activator gene. It does not modify the transmembrane zone.
Patent EP810283A2 describes the construction of an App vaccine strain by modifying the ApxIC gene in such a way that this does not produce the activator protein in a functional form and this can not activate the toxin by acylation. It neither modifies the transmembrane zone.
Jansen et al. (Infection and Immunity 63: 7-37 (1995)) described the production of App homologous recombinants by site directed mutagenesis.
These mutants present the apxIA gene which is inactivated by insertion of the CMr gene and/or the apxIIA gene inactivated by insertion of the TETr gene.
Tascón et al. (Molecular Microbiology 14: 207-216 (1994)) describes two App mutants. One of them has a disruption in the gene apxIBD and the other a disruption in the structural gene apxIA.
Reimer et al; (Microbial Pathogenesis 18: 197-209 (1995)) describes an App. a virulent mutant which, by chemical mutagenesis, has deletions that affect important parts of the operon apxIABCD. This mutant does not synthesize the ApxI toxin, but is able to synthesize the ApxII, although this is not secreted from the cell.
Strains that do not express ApxI and ApxII exotoxins can not be used as attenuated vaccines because they do not induce protective immune responses since the ApxI and ApxII exotoxins are one of the most important virulence determinants of App.
Prideaux (The 16th International Pig Veterinary Society Congress, Melbourne (Australia) 17-20th September 2000, pag. 439-442)) describes a vaccine prepared from a strain with an inactivated apxIIC gene that secretes and express a non-activated ApxII toxin unable therefore to attach to the target cells.
So, the live attenuated vaccines described in the previous background of the invention, based on App strains without haemolytic capability, are less immunoprotective because they have suffered modifications in their structure that do not allow them to attach to the membrane receptor of the target cells. Furthermore these can not generate antibodies against ApxI and/or ApxII toxins, since these are not secreted by the cell. Frey et al. (Gene 142: 97-102 (1994)) describe the amino-acid sequence of the ApxI exotoxine from a serotype I strain and Smiths et al.; (Infection and Immunity 59: 4497-4504 (1991)) describe the amino-acid sequence of the ApxII exotoxin of a serotype 9 strain.